PROJECT SUMMARY Gonadotropin-releasing hormone (GnRH) is a primary and essential regulator of vertebrate reproduction. The decapeptide GnRH is released by GnRH neurons and secreted to the pituitary where the signal is propagated throughout the hypothalamic-pituitary-gonadal axis to regulate reproduction. GnRH neurons originate in the olfactory placode, migrate through the olfactory bulb to the hypothalamus, and project axons into the median eminence where they release GnRH. When GnRH neurons are compromised, GnRH expression is attenuated, producing the condition Idiopathic Hypogonadotropic Hypogonadism (IHH). IHH is characterized by infertility and/or delayed or absent puberty. Although clearly a crucial factor in the reproduction of mammalian species, little is known about the genetic regulators of GnRH neuronal ontogeny. With this research project, I plan to elucidate developmental and genetic mechanisms that control the migration and maturation of GnRH neurons, and therefore illuminate the novel mechanisms responsible for IHH. Through the work proposed under this F31 application, new genetic targets will be identified that could provide novel therapies for treating GnRH deficiency. I propose to study the closely related homeodomain transcription factors, sine oculis-related homeobox 3 (Six3) and Six6, to determine their role in GnRH deficiency. My preliminary data show a profound importance of Six3 and Six6 in GnRH neuron development, survival, and migration. I am proposing 3 aims that will discern the roles these genes play in the regulation of GnRH neurons. The first aim will address the role of the Six3 gene in the development, survival, and differentiation of GnRH neurons, while Aim 2 will focus on the GnRH neuron-specific role of Six6. Aim 3 will study receptors, ligands, and cell-cell communication genes that are responsible for GnRH neuron survival along the migratory route from the olfactory placode to the hypothalamus. To execute these aims, I will utilize both GnRH neuron-specific knock-out mice (Six6Flox/ GnRHCre), and full-body knock-out mice (Six3Het) to investigate the cell autonomous and systemic implication of Six3 and Six6 on GnRH neuron development, survival, and migration. Specific analysis will involve detailed assessment of GnRH neuron survival using immunohistochemistry for GnRH neuron staining, apoptotic assessment, and lineage tracing. I will also determine the contribution of Six3 and Six6 to fertility in the Six6Flox/GnRHCre and Six3Het mice. In addition to these in vivo mouse models, I will explore the contribution of Six3 and Six6 to in several GnRH neuron cell lines. In these in vitro models I will use siRNA for Six3 and Six6 to detail the importance of these genes in GnRH neuron cell survival, migration, and in the expression of known survival cues that delineate the migratory pathway of GnRH neurons. These investigations will enable me to garner knowledge concerning the novel regulatory mechanisms of this fascinating hormone's release; and, will allow us to shed light on causes of infertility plaguing ~7% of all couples.